Traditional Liquid Chromatography, High Performance Liquid Chromatography or paper chromatography can be used to separate mixtures of radio-labelled compounds. This results in the isolation of different fractions whose radioactivity can be measured by mixing an aliquot with about two to four times its volume of scintillation liquid (generally referred to as a "scintillation cocktail") and thereafter counting light events produced by the radioactivity of the fraction. Thus liquid scintillation counting is known to be useful for measuring the concentration of a radio-labelled species in a sample or fraction.
Liquid chromatography has long been known to be a capable technique for the separation and analysis of multi-component mixtures. However HPLC has now progressed to be the method of choice for the quantitation of components which could be thermally unstable and non-volatile. This progress can be attributed to improvements to and development of chemically bonded stationary phases. Also solvent programming (gradient elution) is now commonplace in HPLC. In normal bonded phase chromatography the stationary phase is polar (e.g. Silica) and the mobile phase (elution solvent) is non-polar. In this case the non-polar species in the mixture elute first due to their preference for the mobile phase (i.e. exhibit lower retention). With reversed phase chromatography the stationary phase is nonpolar (Octadecyl Silica) and the mobile phase is polar. The elution order may be the reverse observed with normal bonded phase chromatography. Thus it can be seen that HPLC is an effective method for the separation of multi-component mixtures containing radio-labelled species.
Liquid scintillation counting is widely utilised to analyze samples containing radioactively labelled substances. Typically a sample in solution is mixed with a liquid scintillation cocktail and the light events produced from the sample and cocktail mixture are detected according to their energy and number of events. The light events occur when the energy of the particles, emitted form the radioactive isotope component of the sample in solution, is transferred to the molecules of liquid scintillator. This produces a light emission of a specific energy range which is characteristic of the radioactive isotope.
Detecting both the energy and number of light events in a particular energy range provides the information necessary to construct a spectrum. Using this information the radioactive species can be quantitatively analyzed. Liquid scintillation counting and instruments to perform liquid scintillation counting have been widely discussed in a multitude of publications and patents.
The separation and determination of radioactive species which are present in a multi-component mixture can therefore be accomplished using a separation scheme such as HPLC followed by a radio-assay. Collection of column effluent fractions followed by liquid scintillation counting has been used extensively, but has the disadvantage of being time consuming, requires manual manipulation of samples and compromises resolution.
A continuous flow-through radioactivity detector minimises these disadvantages and several such continuous-flow radioactivity monitors have been described in the literature; see, e.g. D. R. Reeve and A. Crozier (1977) J. Chromatogr. 37, 271, "Radioactivity monitor for high performance liquid chromatography"; E. Schram (1970), "Flow monitoring of aqueous solutions containing weak B-emitters" In: The Current Status of Liquid Scintillation Counting, E. D. Bransome, Jr. M.D. (ed.) Grune and Stratton, New York, pp 95-109; and L. R. Snyder and J. J. Kirkland (1979) In: Introduction to Modern Liquid Chromatography, John Wiley & Sons, Inc., New York, pp 158-161. Other relevant publications include Flow through radioactivity detection in HPLC, (Progress in HPLC volume 3), eds. H. Parvez et al. (1988) VSP B.V. and Radiochromatography--The Chromatography and Electrophoresis of Radiolabelled Compounds, T. R. Roberts (1978) Elsevier.
Prior liquid scintillation cocktails known to applicants for use in flow-through cells for measuring radioactivity in fractions from HPLC procedures have certain drawbacks. These scintillation flow cocktails are based on conventionally used solvents which include toluene, xylenes, cumenes, ethylbenzene and pseudocumene. Other solvents which have been used are mono-, di- and tri-alkyl benzenes.
These solvents suffer from the disadvantage that they have relatively high vapour pressures, relatively low flash points and relatively high toxicity. A further disadvantage is that they are not biodegradable. Although these solvents suffer from the above mentioned disadvantages their relatively low viscosity makes theft suitable as media either alone or in conjunction with alcohol-type diluents for scintillation cocktails usable in flow systems.
The advent of safer solvents suggested that all the disadvantages associated with the conventionally used solvents would be overcome. The safer solvents now in use in liquid scintillation counting include di-isopropylnaphthalene, linear alkylbenzenes and phenylxylylethane and these solvents are characterised by their low toxicity, low flammability, high flash point, low vapour pressure and biodegradability.
These safer solvents have been tried as bases for scintillation cocktails usable in flow systems but they all suffer from having too high a viscosity. The high viscosity causes high back-pressure in the flow system, which can exceed the maximum design pressure of the pump, fittings and even the flow cell.
Another disadvantage of a high viscosity is the inhibition of fast mixing of the scintillation flow cocktail with the HPLC eluent containing the radioactive species, which fast mixing is necessary for an accurate and reproducible determination of the radioactivity. The much higher viscosity of these solvents has therefore precluded their widespread use even after modification with alcohol-type diluents. The amount of diluent necessary to reduce the viscosity to instrument-usable levels results in a substantial reduction in detection sensitivity with respect to counting efficiency.